Summary / Abstract Breast milk is associated with protection against both early childhood illness and lifelong risk of disease in offspring, but the mechanisms by which human milk bestows these protective effects are largely unknown. Intriguingly, the microbiome may represent a key link between breastfeeding practices and disease outcomes, as breastfeeding is independently associated with 1. shifts in the infant gut microbiome and 2. protection against diseases for which the microbiome has been implicated in pathogenesis. For example, necrotizing enterocolitis (NEC), an often fatal inflammatory disease of the neonatal gut, is strongly associated with shifts in the neonatal gut microbiota prior to disease onset, and breastfeeding provides protection against NEC. This suggests that human breast milk may provide protection from disease in part by structuring the infant gut microbiota. Our long-term goal is to characterize bioactive components of breast milk that significantly alter infant gut microbial community structure and bestow protective properties on the infant gut microbiome. The goal of this project is to characterize the impact of two understudied components of breast milk: the milk microbiome and maternal immunoglobulins (IgA). Breast milk contains many putative probiotic species, but its origin and its role in structuring the offspring microbiome have not been well characterized. Additionally, breast milk contains an abundance of maternal IgA which is well-known to provide protection against infections, but its impact on gut microbial structure has not been explored. Our central hypothesis is the maternal microbiome and IgA delivered to the infant via breast milk play a critical role in shaping the infant gut microbiome. We will test this hypothesis in the following three specific aims: 1. characterize the role of maternal diet and gut microbiota in structuring the milk microbiome by using highly-controlled dietary and probiotic intervention studies coupled with culture-independent profiling of the microbiome, 2. characterize the role of the milk microbiome in establishing the gut microbiome of term and preterm infants by comparing the microbiome of breast milk and infant stool samples from mother-infant pairs, and 3. determine the influence of maternal IgA on the offspring gut microbiome using IgA-SEQ, a novel methodology that allows for high-throughput, global characterization of IgA-bound bacterial taxa in the infant gut. Completion of these aims will yield several important findings with implications for early childhood health. Currently, infant formula preparations are not supplemented with probiotics or immunoglobulins, and this research represents a first step in elucidating the importance of these components for establishment of the human microbiome. Overall, the studies proposed herein will provide insight into the importance of breast milk in early development of the microbiome and may eventually inform the design of novel probiotic therapeutics.